1. Field of the Invention
The present invention provides a method for detecting the presence of a retrovirus in a biological sample by detecting the presence of the enzyme, reverse transcriptase. The method utilizes a template and primers in conditions under which a DNA strand is synthesized and subsequently amplified by the polymerase chain reaction, if reverse transcriptase is present.
2. Background Art
Retroviruses are widely distributed in vertebrates and are known to cause a variety of diseases in man and animals including immunodeficiencies, leukemias and lymphomas (1). The entire retrovirus family is characterized by the presence of a unique enzyme, reverse transcriptase (RT), which transcribes the viral genomic RNA into a double-stranded DNA copy (1). This feature has led to studies of the unique enzymatic function of RT for two main applications. First, the presence of RT has been the basis for diagnosis of retroviral infection and for the generic detection of the presence of retroviruses in cell cultures and in the infected host. Second, the RT enzyme constitutes a primary target for antiviral drug intervention (1,2).
The polymerase function of RT from several retroviruses has been well characterized and shown to require an RNA template, a primer, triphosphate, a divalent cation and physiological salts (1). Therefore, assays for RT activity have conventionally used these reagents and conditions to measure the ability of a sample to produce a DNA copy of a known exogenous RNA template (e.g., poly rA) by synthesizing a complementary DNA oligonucleotide primer with radiolabeled nucleotides (e.g., .sup.3 H- or .sup.32 P-dTTP). RT synthesized DNA has been detected by measuring incorporation of the labeled nucleotide (3,4) and more recently, by assays which employ nonradioactive nucleotides in enzyme linked immunosorbent assay (ELISA) formats have been developed (5-8) and by polymerase chain reaction (PCR) (17), as further described below.
Although RT assays continue to be an essential laboratory tool for the identification of known and novel retroviruses (4-11), the successful use of RT assays has been limited to the detection of retroviral particles in culture supernatant (4-8), which has the disadvantage of requiring that a virus be cultured before detection. By the present methods, lentiviruses, such as human immunodeficiency virus-1 (HIV-1) may be readily detected, but oncoviruses, such as human T lymphocytic virus types I and II (HTLV-I and HTLV-II), are much more difficult to detect, presumably because of poor RT activity and because they are typically cell-associated, which means their RT is less accessible for detection in culture supernatants (6-8). This limitation has rendered RT testing of little value in the detection of HTLV infection.
Despite multiple attempts to improve the sensitivity of RT assays, the direct detection of retroviruses in clinical samples (e.g., serum) has been unsuccessful (4-12). For example, in studies of HIV-1 infected individuals, detection of virus in plasma by RT assays has been largely abandoned because of the low sensitivity of this method (12).
The inability to detect RT activity in serum hinders the use of this virological marker in diagnosing disease, monitoring drug efficacy in patients, monitoring virus load and predicting disease progression. Present methods for qualitative and quantitative detection of plasma HIV-1 include viral isolation and p24 antigen capture (13-15). However, these assays have disadvantages. Virus isolation from plasma requires virus culture that is typically maintained for 14 to 28 days and is labor intensive, time consuming, fraught with biological variation and is not a universal marker in the infected population given the low levels of HIV-1 in patients with CD4+ T lymphocyte counts of &gt;200/mm.sup.3. Similarly, p24 antigen, either free, virion-associated, or immune complexed, is not always present in the HIV-1 infected population.
RT-PCR permits the qualitative detection of the cell-free virus in plasma using an exogenous RT and a primer pair of known sequence to amplify viral RNA sequences in the plasma (15,16). Although RT-PCR has been reported to be highly sensitive, this assay requires RNA extraction and multiple sample manipulations that may increase the risks of PCR contamination. The RT-PCR assay may be complicated further by the lack of a standardized universal quantitative test and the variabilities that may be incurred during processing and storage with regard to the degradation of the genomic RNA. Although RT-PCR, like antigen capture, is highly specific, a knowledge of the nucleotide sequence of the target retrovirus fragment is necessary for primer development. Given this limitation, RT-PCR is not suitable for detecting variant, novel, or unknown retroviruses.
Recently, two assays for RT, which use the RNA of bacteriophage MS2 or brome mosaic virus (BMV) as a template and PCR as a detection system, have been reported to be highly sensitive for the detection of murine leukemia virus RT and other stocks of retroviruses in serum (18,19). However, the evaluation of the specificity and the sensitivity of these assays on adequate numbers of serum specimens was not disclosed (18,19). In addition, in both of these assays, problems with inhibitors of RT activity in serum and nonspecific background RT activity have been described (20), raising serious questions about the diagnostic value of these assays.
This invention provides a RT assay that employs a PCR-based amplification system to detect a known cDNA product of the RT reaction. The assay of the present invention, referred to hereafter as Amp-RT, is highly sensitive and specific, requires no knowledge of viral genomic sequence and allows the detection of RT activity in samples of individuals infected with retroviruses or any other biological entity that produces RT.